Recombinant ferritins for multimodal nanomedicine

Abstract In all living organisms, ferritins are a group of proteins important for maintaining iron homeostasis. Increasing amount of studies has shown that recombinant ferritins can be widely used in multimodal nanomedicine, especially for anticancer treatment and vaccination. Recombinant particles prepared by fusing viral proteins and ferritin subunits produce a better immune response and higher antibody titres. Moreover, actively-targeted ferritin nanoparticles can recognise receptors and deliver natural or chemical drugs specifically to the tumour tissue. In addition, ferritin-linked or loaded with contrast agents or fluorescent dyes can be used as multimodal particles useful cancer theranostics. In this review, we fully summarised the unitisation of recombinant ferritins in multimodal nanomedicine. The research progress of using recombinant ferritins as nanovaccines, nanozymes, and bioengineered nanocarriers for targeted therapy and bioimaging is emphasised.


Introduction
Ferritins (Ftn) are a class of important iron storage proteins widely present in all organisms that use Ftn for maintaining iron homeostasis 1 . Ftns usually consist of a spherical cage of 24 identical or heterologous H-and L-subunits 2 . Of these, the H-subunit provides rapid detoxification of iron through its inherent ferroxidase activity and the L-subunit promotes iron nucleation, mineralisation, and long-term storage 3 . As a natural polymeric biomaterial, Ftn has the advantage of good biocompatibility, biodegradability, and a long plasma half-life 4 . Noteworthy, Ftn is an important disease biomarker. Serum Ftn is routinely used in clinical medicine as a surrogate indicator of iron storage in the body, where low levels indicate iron deficiency 5 . Also, an inflammation or infection can dramatically alter serum Ftn levels 6 . In the pancreas, Ftn deposits can impair cells function and induce diabetes. Moreover, it can also lead to hypogonadism and hyperpigmentation in the pituitary gland and skin, respectively 7 . There are some hereditary ferritinopathy and neuroferritinopathy associated with iron accumulation, such as amyotrophic lateral sclerosis 8 , restless legs syndrome 9 , and Alzheimer's disease 10 . High levels of serum Ftn are a marker of poor prognosis in restless legs syndrome.
With the development of recombinant DNA and protein engineering technologies, recombinant proteins have also shown unique potential for use in the biomedical research 11 . In recent years, there is a growing interest in Ftn-based vaccines 12 . The reversible assembly of subunits of Ftn using gene fusion SARS-CoV-2 vaccine 13 , which is in phase I clinical trials 14 . In addition, HIV and influenza vaccines have also been successfully designed [15][16] .
Besides use of Ftn in vaccination research, removal of the iron from Ftn internal cavity results in a hollow Ftn cage structure that can be loaded with variety of bioactive compounds and possesses many advantages as a carrier for cancer-targeted drugs 17 . For example, Ftn loaded with doxorubicin (Dox) finctionalized with polyethylene glycol (PEG) preferentially crossed the airway mucus barrier and released Dox uniformly into the orthotopic lung tumour tissue, increasing the survival rate of mice by up to 50% 18 . Additionally, loading of siRNA effectors into Ftn effectively prevents their degradation and avoids multiple drug administration 19 . Hepatocellular carcinoma-targeted Ftn (HccFtn) can effectively load six times the dose of free Dox used Carcinoma-targeted Ftn can slowly released and showed harmless to the healthy hepatocytes 20 . Moreover, Ftns can be employed as promising modalities in the rapidly developing filed of nanozymes. Engineered recombinant Ftns are an ideal tools to protect artificial metalloenzymes from harsh and complex in vivo environments, as has been shown in case of metallozymes loaded into Ftn. The loading did not alter peroxidase activity of metallozyme and improved generation of intracellular oxygen, thus proved useful for nanozyme-based anticancer therapy 21 . Although intratumoral administration of Ftn exhibits some advantages, uncontrollable proper assembly between recombinant protein subunits and heterogeneity of particles lead to limited clinical application of recombinant Ftn.
In this manuscript, we systematically reviewed the functional structure of Ftn, the research progress and future prospects of recombinant Ftn used in multimodal nanomedicine. We particularly focus on the research progress in the field of nanovaccines, nanozymes, bioengineered nanocarriers for targeted drug delivery and diagnostic imaging. We provide examples of selected applications of Ftn and summarise the advantages of Ftn in the field of nanomedical research, with the aim of providing a reference for the current and future medical research on recombinant Ftn.

Ferritin utilisation in nanovaccine
The modification of the different subunits of Ftn nanoparticles by genetic engineering not only allows the introduction of antigens onto the surface of Ftn, but also ensures a stable and autonomous loading of various types of cargoes into the cavity of Ftn 22 . Using Ftn as vehicle, various vaccine candidates against different types of pathogens are currently in clinical trials H2 influenza 23 , or SARS-CoV-2 vaccines 24 .

Ferritin induces high concentrations of neutralising antibodies
Ftn-based nanoparticles designed for vaccination against SARS-COV-2/S 25 , influenzas and respiratory syncytial virus (RSV) induced high levels of specific antibodies in animals 26 (Figure 1). A/Singapore/1/1957 haemagglutinin (HA) genetically fused to the Helicobacter pylori-derived Ftn (HP-Ftn) was shown to induce a broadly neutralising antibody response against influenza type 1 viruses (including seasonal H1 and avian H5 subtypes) 23 . Furthermore, novel Coronavirus fusion Ftn was used to induce the production of neutralising antibodies in mice, and the induced antibody levels were twice higher than those of plasma donors in the recovery phase of SARS-COV-2 24 . Furthermore, the fusion of HP-Ftn with Epstein-Bar virus (EBV) surface glycoprotein can stimulate mice and primates to produce specific neutralising antibodies higher than those induced by an EBV glycoprotein only 27 . The fusion Ftn nanoparticles produced by fusing the HP-Ftn with the H1N1 influenza virus HA induced a rapid production of neutralising antibodies in mice that were more than 10-fold more potent than those produced by the normal vaccine and even were able to neutralise different subtypes of the influenza virus 28 . As the H-and L-chains of Ftn can be combined with different antigens to prepare recombinant vaccination particles containing two or more antigens, Ftn also enables facile development of multivalent nanovaccines 29 . Staphylococcus aureus (SA) alphahemolysin is a frequent cause of deleterious cytotoxic effects of SA infections 30 . Therefore, a synthetic DNA encoding Hla121-138 was fused to the CDS of Ftn at the N-terminus, cloned into the pET-28A plasmid and introduced into BL21 (DE3) cells to express an epitope-based nanoparticle (EpNP) that immunologically induced potent haemolytic neutralising antibodies and conferred significant protection in a mouse model of SA skin infection 31 .
Additionally, preS1 is an antigenic protein on the surface of HBV. preS1 on the surface of recombinant Ftn actively targets SIGNR1þ macrophages and SIGNR1þ dendritic cells in lymph nodes and promotes the activation of B cells and T cells 32 ( Figure 2). Importantly, it is worth to note that Ftn can stimulate bone marrow dendritic cell maturation via the activated TLR4/NF-jB pathway, which in turn promotes T cell proliferation and differentiation. ct of Ftn nanovaccines may also be related to repeatedly display the target antigen 32,33 .

Molecular size and shape affect the efficacy of recombinant protein vaccine
The immunological effect of antigenic peptides is related to their molecular size and shape. For example, when the RSV protein of enterovirus was presented the surface of Ftn, the antibody titres induced in mice were higher. Within a certain range, the immune effect increases with the antigenic peptide molecule 34 . H. pyloribullfrog hybrids carrying residues 2-9 at the N-terminal end of bullfrog L-chain Ftn allow the formation of radially prominent tails that improve antigen presentation in many Ftn-based vaccine candidates and further enhance the effectiveness of immunisation 13 .
Viral stinger protein particles are recognised by immune cells to induce specific antibody production. By gene fusion techniques that allow the HA gene to be located just at the N-terminus of Ftn around the triple fold axis, HA monomers interact and are stable and allow conformation-dependent oligomerization of the trimeric antigen 35 . In particular, the Receptor binding domain (RBD) of the S-protein structural domain is directly involved in host receptor recognition, and amino acid variants in this region lead to changes in the genophilic and infectious properties of the coronaviruses, like SARS virus, enters cells by recognising the human angiotensin 2 (ACE2) protein of the human host 36 . RBD-based subunit vaccines were designed to provide the basis for new vaccine therapies against the new coronaviruses (nCoV) 12,37 , entry is achieved by binding of coronavirus spike proteins to human ACE2 receptors. Spike-ACE2 interactions are mediated through the receptor RBD of spike proteins 38 . A fusion protein of H1 HA stabilized-stem on Ftn nanoparticle (H1-SS-NP) can induced passive transfer of immunoglobulins protected mice from lethal doses of H5N1 virus killing 13 . The Ftn vaccine induces long-term antibodies with good results. three potentially linear sequences in the Yanaka strain of CDV HA coupled to the Ftn form are known as YaH3F, YaH4F, and YaH5F, respectively 39 . BALB/C mice were immunised subcutaneously with each of the three protein-coupled forms and nucleic acids (mRNA), and serum IgG antibodies increased significantly after 28 days, with higher antibody titres in the protein group than in the mRNA group, even maintaining higher antibody titres after 42 days.
The above-mentioned studies show that Ftn nanovaccines can stimulate both B-cell and T-cell activation, thus inducing a complete immune response. Besides, Ftn itself can be used not only as a non-viral vaccine transport carrier, but exhibits also a good effect in enhancing the immunity itself. Self-assembled nanoparticles designed by altering the structure of Ftn induce broader and more effective immunity than traditional influenza vaccines. Protocols need to be standardised so that the biological process of production becomes reproducible and Ftn-based therapies become relatively accessible. Because most self-assembled sequences are derived from species other than humans, one problem with self-assembled protein nanoparticles as vaccines is that off-target immune responses 40 . If they occur, they may limit, to a greater extent, the effectiveness of subsequent booster immunizations or other vaccinations using vaccines containing the same self-assembled structural domains. The mechanism by which Ftn nanovaccines induce a high level and sustained immune response was found to be deposition of Ftns in the lymph nodes of immunised mice and stimulation of specific B lymphocyte maturation; however, this phenomenon was not found in immunised macaques 41 . Thus the specific mechanism of sustained immune response to recombinant Ftn vaccines needs to be further investigated.

Ferritins as artificial nanozymes
Nanozymes are a class of nanomaterials that exhibit intrinsic enzymatic properties and possess enzymatic catalytic properties at the nanoscale that only natural enzymes have 42 . Under mild conditions, artificial nanozyme has certain reaction kinetics 43 . Ftn is a natural nanozyme, which shows intrinsic enzyme-like activities (iron oxide enzyme, peroxidase) and first to report artificially prepared ferromagnetic nanoparticles with peroxidase-like activity 44 . Biomimetic synthesis of nanozymes in Ftn shell and full use of self-assembled nanocage structure can effectively combine the advantages of self-assembled Ftn nanocage and nanozyme. In tumour therapy and chemotherapy, hypoxia has always been the key factor affecting drug resistance. Therefore, reducing O 2 consumption of tumour mass is one of the ways to alleviate tumour hypoxia 45 , which can increase pO2 in cancerous tumour tissues, improve the hypoxic microenvironment of tumours, restore the sensitivity of tumour cells to chemotherapy and radiotherapy, and thus generally improve the therapeutic effect. What's more, Ftnloaded enzymes also have good stability, remaining biologically active after high temperature and detergent treatment. Thus, the peroxidase activity of Ftn could have a promising applicability in tumour therapy ( Figure 3).

Ferrtin is used as a carrier for nanozymes
Transferrin is a receptor on the surface of the cell membrane, which can specifically recognise Ftn 46 . CD71 is the most common transferrin receptor, which has been shown to allow encapsulation of an artificial transfer hydrogenase (ATHase) based on streptavidin-biotin technology which catalyses the reduction of cycloimine detoxification after the artificial transaminase has also been encapsulated in the cavity of horse spleen apoFtn (HSAF) 47 . HSAF provides a protective protein shell for precious metal-based artificial enzymes to defend against and adapt to complex physiological environments and deliver them to targeted sites in the body. There are also some advantages in transporting other substances through the modification of Ftn.
In addition to spontaneous formation of iron oxide core nanoparticles, the Ftn cavity can also accommodate a diverse range of artificially prepared metal nanoparticles. Synthesis of platinum nanoparticles in the cavity of desferrin in vitro, protein-platinum nanoparticles showed good catalytic efficiency and long-term stability. Subsequently, after the Ftn-receptor-mediated uptake into human intestinal Caco-2 cells, the catalytic activity of the particles was tested. Under externally induced pressure, intracellular concentration of hydrogen peroxide decreases, and cell viability increases 48 . Materials with four enzyme-like activities responsible for reactive oxygen species regulation (oxidase, peroxidase, catalase, and superoxide dismutase) developed with nitrogen-doped porous carbon nanospheres were introduced into Ftn into the lysosome and promoted the production of reactive oxygen species in a tumour-specific manner, leading to significant tumour regression in a mouse model 21 . Therefore, Ftn nanozymes are powerful tools capable of regulating intracellular reactive oxygen species. Loading into Ftns is hence a promising strategy and nanozymes targeting tumour cells can be constructed not only for in vivo anticancer applications but also for other biomedicinal purposes such as antiatherosclerotic therapy 49 . Because active macrophages infiltrated by plaques drive atherosclerosis, plaques are more prone to breakage and show a dominant correlation, so HFtn-Fe 3 O 4 nanozyme can specifically stain unstable ruptured plaques. AgNP obtained in pfFtn is used in areas such as in vivo diagnostics, antimicrobial or oncological therapy 50 .
Ferrtin nanozymes are resistant to high temperature Furthermore, palladium and silver nanoparticles (NPs) anchored on the outer surface of Ftn form stable suspensions of uncoated particles with multiple catalytic and mimetic enzymatic activities. The nature of pfFtn-Pd Ftn is relatively stable, detergent and an optimum temperature of about 80 C, high temperature will not destroy the enzyme activity, silver nanoparticles were detected in the presence of sodium dodecyl sulphate (SDS) before mimicking enzyme peroxidase activity, and under this condition, most of the natural enzymes were denatured quickly, while pfFtn-Pd Ftn nanozymes still maintained high stability and catalytic activity due to the excellent thermal stability of pfFtn, showing strong adaptability 51 (Figure 4).
Ftn nanozymes are an ideal strategy to protect the catalytic activity of artificial metallic nanoparticles from harsh and complex environments, allowing for the delivery of artificial metalloenzymes in living organisms. Artificial nanozymes have the advantages of high biocompatibility, low reaction toxicity, controllable synthesis, and targeted modification, but the disadvantages are that their types are limited. Technology of preparation of nanozymes is still immature, and they are rarely working in living organisms. However, nanozymes field is rapidly advancing and at present, Ftn nanozymes have been successfully tested in certain applications in the medical field. The appearance of Ftn nanozymes also inspires us to learn from natural nanostructures (such as R-chymotrypsin, b-Gal, b-lactamase) to optimise or design nanozyme reasonably.

Ferritin as bioengineered nanocarrier
Nanocages are good transport materials after Ftn removal of the iron core. Anticancer drugs need to be delivered into tissues and cells with a high level of selectivity and fidelity. Chemotherapy is the main treatment for cancer and despite killing cancer cells; the high levels of the drug can affect the entire network of cells in the body and produce serious side effects 52 . Hence, in the process of delivery, we should not only consider their biocompatibility, toxicity, immunogenicity, and therapeutic index, but also comprehensively consider the disadvantages of the developed nanocarriers, such as non-specific distribution, rapid clearance, uncontrolled drug release, and low bioavailability 53 . However, Ftns can be used specifically to deliver chemotherapeutic drugs and is effective in circumventing these risks. The advantages of the Ftns is the ease of surface modification, high loading capacity, and high biocompatibility making Ftn a multifunctional nanocarrier for the research and development of targeted drugs carrier 54 . Ftn is highly expressed in tumour-associated macrophages, and its down-regulation can destroy the supportive tumour microenvironment, kill cancer cells and increase the sensitivity to chemotherapy 55 . Besides, human heavy chain Ftn (HFtn) can be specifically recognised and internalised by the CD71 56 , which is highly expressed in most tumour cells, thus facilitating effective drug-loaded Ftn cellular uptake [22][23] . Ftn has made significant progress in drug encapsulation techniques, starting from single drug encapsulation to two-drug mixed encapsulation and now regional encapsulation, which is highly appealing 57 .

Recombinant ferritin loads with a single substance to treat various diseases
Hepatocellular carcinoma (Hcc) is one of the main causes of cancer-related deaths, accounting for the second most common in the world 58 . GRP78 was identified as the membrane receptor of Hcc-targeted peptide SP94 59 , which was displayed on the surface of Pyrococcus furiosus Ftn 20 . GRP78-targeted Ftn nanocaged can carry ultra-high dose of Dox for Hcc therapy. Dox encapsulated in HccFtn-Dox was selectively delivered to Hcc cells and effectively killed subcutaneous and pulmonary metastatic Hcc at more than 10-fold higher average levels than those reported for Ftn-based nanocarriers without the GRP78 targeting peptide 20,60 ( Figure 5). In addition, HccFtn-Dox reduces drug exposure and damage to the liver and kidney, which are the main organs of drug metabolism 21 .
CD71, a transferrin receptor that specifically recognises Ftn, was utilised to deliver Ftn-encapsulated Cyt C to an acute promyelocytic leukaemia (APL) NB4 cell line, resulting in apoptosis 46,61 . This trial was one of the pioneering studies to employ Ftn assembly for Cyt C transportation. In a subsequent in vivo study, the surface of the human HFtn nanocage was further functionalised with polyethylene glycol (PEG), which facilitated effective penetration of the mucus barrier and tumour tissue 18 . Using CaP wrapping can effectively shield and protect Ftn, when transported to the weak acid environment of the tumour, and thus release the drug after binding to CD71 into cancer cells, promoting tumour calcification and immune response 62 . In addition, human HFtn can encapsulate different types of drugs within its lumen, can bind to the receptor CD71, and the novel nanodrug-0504 contains about 80 molecules of a potent, broad-spectrum, non-camptothecin topoisomerase I inhibitor (Genz-644282) and can be produced industrially in a pure and homogeneously formulated product which can belyophilized 63 .
Encapsulation of Genz-644282 with HFtn showed excellent therapeutic efficacy in an in vivo pancreatic cancer model (Figure 6), significantly increasing the overall survival rate of the animals 64 .
What's more, paclitaxel (PTX) is a chemotherapeutic agent, limited in the use by its lack of targeting and insolubility in water 65 . Natural human HFtn nanocages can be used as a carrier for PTX  to form HFtn-PTX for targeted delivery, which can bind tumour cells by interacting with CD71, leading to endocytosis, and apoptosis assays show that HFtn-PTX has similar apoptotic characteristics to free PTX for MDA-MB-231 cells 66 . The fusion of the penetrating peptide tLyP-1 with the N-terminal end of human HFtn and wraps more PTX molecules in HFtn by endocytosis to form tLyP-1-HFtn-PTX. tLyP-1-HFtn-PTX exhibits greater cytotoxicity and anti-invasive ability, good permeability, and growth inhibition against SMMC-7721 spheroids. Animal experiments on BABL/c nude mice focussed on selective accumulation and penetration into the tumour mass in vivo showed higher therapeutic efficacy and lower systemic toxicity 67 .
HFtn can be targeted by recognising CD71, absorbed through endocytosis and then degraded by lysosomes, and epitope-modified HFtn can further show better efficacy and potential 46 . Curcumin loading can be used in the same way, with curcumin encapsulated in HFtn to make CHFtn, which inhibits both MDA-MB-468 and MDA-MB-231 cell lines of triple-negative breast cancer (TNBC), suppressing both at G2/M and G0/G1 stages, respectively, and can be used in TNBC treatment 68 . Similarly, the increased cellular uptake and anticancer activity of geftinib-loaded HFtn was found in breast cancer (SKBR3) cells. Compared to HFtn alone, HFtn loaded with gefitinib exhibits sustained pH-responsive release of gefitinib, allowing effective delivery of gefitinib to breast cancer cells 69 .
Till now, more than a dozen drug molecules were successfully introduced into the Ftn nanocages, indicating that the one-step method is a powerful vehicle for the construction of drug-loaded Ftn delivery systems 70 . Human HFtn has a natural two-fold axis loading channel with an average diameter of 0.9 nm 71 . In addition, the open state of this drug entry channel is sensitive to temperature changes between 4 C-37 C, the natural drug channel is closed when incubated at 37 C-65 C, the amount of Dox loaded into HFtn gradually increases and reaches the peak at 65 C, and can stably encapsulate about 90 Dox molecules 71 . Temperature directly determines the opening and closing of such channels 72 , as 30 mM urea can expand the Ftn native channel size 73 . Loading Dox through channel into HFn nanocages allows for higher drug loading efficiency, higher HFtn recovery, and better stability 71 . Moreover, channel-loaded HFn-Dox has excellent biosafety and significantly improved antitumor activity 71 . For this purpose, the mixture was incubated at the appropriate pH to obtain complexes of Ftn and the target drug. The electrostatic potential facilitated the passage of drug molecules through the pores, across the Ftn shell, and the deposition of drug within the Ftn cavity. The constructed Dox-HFtn loading was more than 3 times higher than the free dosage, and the recovery of Dox was improved by 10 times 74 .

Ferritin as a carrier of photothermal and photosensitizers for cancer therapy
In recent years, Ftn has been shown to play an important role in phototherapy as a carrier of photothermal agent and photosensitizer and can realise the combined treatment of chemotherapy and phototherapy 75 . Photothermal therapy (PTT) with Ftn cages is a new direction in targeted therapy, such as encapsulation of copper Ftn followed by laser irradiation, which kills 100% of human glioblastoma cells and without obvious toxic side effects 76 . Prussian blue (PB)-modified Ftn nanoparticles (PB-Ft NPs) have peroxidase activity, which can catalyse the oxidation of chemotherapy drug 3,3 0 ,5,5 0 -tetramethylbenzidine by hydrogen peroxide into oxidised TMB, and can successfully inhibit the growth of murine breast cancer cell line (4T1) 77 . Ftn modified by CGKRK polypeptide was constructed by genetic engineering. Fluorescence intensity test confirmed that targeting CGKRK peptides modified Ftn maintained ROS-producing ability. Photodynamic therapy (PDT) is a widely known oncology therapy that can be combined with PTT to treat tumours. In the PTT þ PDT group, the tumour subsided, and the tumour did not recur after 16 days of treatment, indicating the advantages of synergistic treatment 78 . PB-Ft nanoparticles are a good sensitiser for tumour chemotherapy. PTT mediated by PB-Ft NPs may have the ability to reverse drug resistance. Folic acid was coupled to the surface of Ftn as a tumour-targeting ligand, then Ftn nanocages were subsequently loaded onto ZnF16Pc. The resulting recombinant Ftn nanoconjugates could effectively inhibit 4T1 tumours in vivo and effectively inhibit tumour growth and tumour metastasis in mice under light exposure 79 .

Ferritin delivers nucleic acid-based drugs
The use of recombinant Ftn for polypeptide and gene delivery can also regulate molecular release. Nucleic acid-based drugs, including small interfering RNAs (siRNAs), are becoming increasingly important due to their remarkable efficiency in the treatment of various diseases 80 . The siRNA can be encapsulated in unmodified human Ftn. The encapsulation of siRNA into the Ftn is not dependent on the sequence of the siRNA, and the siRNA is protected from degradation, resulting in efficient delivery into human tumorigenic cells, human primary mesenchymal stem cells, and peripheral blood mononuclear cells 81 . At the siRNA concentration of 10 nM, the high-efficiency silencing of the target gene can be achieved by using Ftn in tumour cells and PBMCs as a delivery agent, immune activation of human T cells and B cells is not induced, and the internal anti-inflammatory effect is displayed, indicating that human Ftn can be used for siRNA delivery under the condition of infection or inflammatory disease.
The choice of phosphorylated Ftn nanocage avoids these risks and has a high drug encapsulation rate 82 . What's more, in the simulated gastrointestinal tract test, Ftn can slow down the release rate of procyanidins (PCS) 83 . In addition, Ftn has been also used to improve water solubility, stability, and cell uptake efficiency of rutin and epigallocatechin gallate 84 .

Ferritin nanocarriers can carry two drugs simultaneously
The fusion of the C-end of the human HFtn subunit with an optimised hydrophobic peptide resulted in a modification of the inner surface of the Ftn cavity 85 . This modified cavity was able to simultaneously load Dox and striline, leading to significant anti-tumour effects in animal models of neuroglioma, metastatic liver cancer, and drug-resistant breast cancer 85 . Curcumin and quercetin have been shown to have anti-tumour properties by blocking different signalling pathways 86 . These two bioactive substances are coencapsulated within Ftn, which enhances their synergistic cytotoxicity by increasing the loading rate and targeting cancer cells 86 . Shrimp Ftn can separate chlorogenic acid and b-carotene through reassembly and emulsification 87 . In addition to its potential in treating common tumour diseases, ATP-loaded Ftn has also shown promising in treating asthenospermia by crossing the blood-testis barrier 88 . Therefore, the use of Ftn encapsulation can reduce drug clearance, extend the half-life, reduce drug dosage or liver toxicity. Ftn can serve as a promising drug or nutrient carrier for delivery in vivo.

Ferritin as bioimaging tool
A bone-targeting peptide that specifically binds osteoblasts and hydroxyapatite was fused to the N-terminal part of the Human HFtn, and the hybrid Ftn nanoparticles were labelled with fluorescent dyes. Upon binding of the modified Ftn to osteoblasts and hydroxyapatite specific fluorescent signals were observed in the lower limbs, indicating that recombinant Ftn nanoparticles could specifically recognise the bone tissue, and in the future, Ftn could be applied in clinical treatment of bone diseases 89 (Figure 7). Bifunctional carbon dots (CDs) attached to the surface of HFtn interacting with Dox emitting bright red fluorescence can be used for sensitive in vivo bioimaging 90 . Despite the potential of iron oxide nanoparticles (SPIO) as a blood contrast agent, medical applications remain limited due to their short half-life of a few hours. The contrast agent prepared by temporarily opening the pores of the red blood cell membrane and encapsulating SPIO nanoparticles in red blood cells (RBCs) has a long half-life, which can be used as a magnetic resonance imaging (MRI) contrast agent. Due to the short half-life of standard superparamagnetic iron oxide (SPIO) in vivo 91 , at 2.5 mg Fe/kg, standard SPIO is only stable for 7 days for MRI bioimaging of the liver in mice, whereas deferrin iron in 45 days still can be detected 92 . Terahertz (THz) bioimaging has attracted much attention due to its ability to acquire physicochemical information in a label-free, non-invasive and non-ionizing manner. To date, a variety of macroscopic biological samples (such as burned skin, brain tissue, and various cancerous tissues) have been imaged using THz imaging, with graphene chosen as the substrate for terahertz presentation 93 . Ftn can be used for disease diagnosis and provide a non-invasive and highly sensitive imaging method. By graphene-mediated of THz scattering type scanning near-field optical microscopy, Ftn and IgG can direct imaging 94 . The THz direct imaging of individual biomolecules using Ftn opens up a new avenue for imaging individual biomolecules by coupling effects to real-time monitoring and does not require repeat injections.
Besides, bone marrow mesenchymal stromal cells (BMSCs) were transduced with a lentivirus containing a shuttle plasmid carrying the Ftn heavy chain 1 (Fth1) gene, and the migration and proliferation of mesenchymal cells in a rat stroke model were monitored long-term using magnetic resonance imaging (MRI) of Ftn transgene expression 95 . Ftn expression in stromal cells was evaluated with western blotting and immunofluorescent staining. Bone marrow mesenchymal stromal cells (BMSCs) were transduced with a lentivirus containing a shuttle plasmid (pCDH-CMV-MCS-EF1-copGFP) carrying the HFtn gene. After middle cerebral artery occlusion surgery, Fth1-BMSCs and superparamagnetic iron oxide (SPIO)-labelled BMSCs were injected intravenously into the internal jugular, and monitoring imaging and signal intensity by observing in vitro and in vivo revealed that MRI signal intensity of SPIO-BMSCs gradually decreased over time and that Prussian bluestained cells were present around and in the centre of the infarct area in both transplantation models. Fth1-BMSCs transplanted for the treatment of focal cerebral infarction was safe, reliable, and traceable by MRI. Fth1 marker was more stable than the SPIO marker and suitable for long-term follow-up.
The application of recombinant Ftn is not only in disease diagnosis, but also in the detection of harmful substances. Methylmercury (MeHg þ ) accumulates mainly in the brain and therefore in vivo imaging detection of MeHg þ in the brain is crucial 96 . By integrating the bioimaging of gold nanoclusters (Au NCs), the fluorescence of Au NCs quenched by MeHg þ , and the brain-targeting function of the constructed 16-mer shell-like Ftn (7 A), the prepared 7 A-Au NCs were found to be not only suitable for bioimaging of BBB endothelial cells, but also an effective probe for brain-specific bioimaging MeHg þ detection. The recombinant Ftn has the advantages of low toxicity, high sensitivity, and long-term presence in the human body, no need for repeated injections, and easy detection as a bioimaging material. In the future, Ftn will play an important role in the clinical diagnosis of human diseases and detection of harmful substances (Table 1).

Concluding remarks and perspectives
Based on the suitable immunogenicity of recombinant Ftn, various recombinant protein vaccines have been developed by fusing genes of different pathogens including HIV, SARS-CoV-2, and influenza. Ftn nanozymes show promise for the treatment of cancer, atherosclerosis, and diagnostic. The advantages of Ftn nanozymes are stability, controlled size synthesis, and the ability to be further modified. As targeted cancer drugs, Ftn delivers PTX to mammary cancer tissues, and also reshapes the tumour immunosuppressive microenvironment, and activates intrinsic and adaptive immunity, leading to durable immune memory and anti-tumour effects. In addition, Ftn has been developed as a drug delivery vehicle for hundreds of drug delivery systems. Recombinant Ftn is a bioimaging material that persists in the body and can be used for liver diagnostics, brain infarction detection, and the detection of MeHg þ . Ftn nanoparticles are biocompatible with almost all living organisms, the raw materials are cheap and easy to obtain, the preparation process is economical and environmentally friendly, the structure of the product is well defined, the chemical properties are relatively stable and it is easy to degrade and metabolise in vivo. In addition, in vitro recombinant Ftn is simple to synthesise, easy to modify, and easy to implement. These advantages make it very useful for clinical applications. Due to the unique autoloading ability, numerous different types of molecules can be successfully loaded onto Ftn, especially for the loading and targeting of a wide range of active substances, showing its great potential as a nanocarrier in the fields of disease diagnosis, drug delivery, and bioimaging. Currently, nanomaterials are widely used in medicine, pharmacy, and bioassays. However, there are still some issues concerning the development of Ftn nanoparticles that need to be addressed. for example, the immunogenicity of human-derived Ftn and heterologous Ftn vaccines in different mammals or primates are not fully explored and need to be studied. As carriers for loading drugs, Ftn subunits with different loading ranges for target proteins or peptide chain sizes should be studied. The metabolic pathways of heterologous Ftn nanocages or their derivatives in vivo are not still clear. The encapsulation rate and drug loading capacity of Ftn for different drugs should be tested. There are still no systematic studies on the changes in various properties of Ftn after modification, so the safety of Ftn surface modification is open to debate and observation. The modification of the contact surfaces between Ftn subunits controls self-assembly, but self-assembly at the threedimensional level still needs to be studied in depth. The selfassembled structures of different subunits allow for reactions with a certain degree of stochasticity and uncertainty, as well as the influence of spatial site resistance effects, which require precise control of the reaction conditions to obtain more satisfactory results. In addition, Ftn nanomaterials still present some problems in vivo, mainly including oxidative stress, inflammation, and genotoxicity generated by iron ions, which at the cellular level can cause the malfunction of major organelles such as autophagosomes, lysosomes, endoplasmic reticulum, and mitochondria. Therefore, due to the complex design of Ftn nanoparticles, it is difficult to meet the criteria for targeted drug delivery and clinical applications still need time.
The development prospect of recombinant Ftn nanoparticles is to expand their physiological functions through surface-modified materials while ensuring their high biocompatibility and biodegradability, in order to develop more products adapted to clinical conditions. It is important to note that the Ftn modification strategy to be used depends primarily on the specific function and the specific goal that the modification gives to the Ftn, rather than simply pursuing its multifunctionalisation. The combination of biological and chemical modification of Ftn surfaces allows for site-specific modifications and the attachment of various macromolecules, including affinity markers, antibodies, fluorescent, glycans, nucleic acids, and target peptides, giving Ftn new properties and significantly broadening its range of applications. Therefore, combining chemical and biological modifications to complement each other's strengths and weaknesses to achieve the modification and multifunctional modification of Ftn is one of the important directions for future development. It is believed that as the research progresses and technology advances, more recombinant Ftn nanoparticles with more diversified structural composition, more intelligent and comprehensive functions will be used in clinical medical research, and Ftn will have more widespread biomedical applications and show its good application prospects.